Internalisation of DNA, using conjugates of poly-l-lysine and an integrin receptor ligand

ABSTRACT

Composition comprising DNA associated with a polycation moiety wherein the polycation moiety is itself coupled to an integrin receptor binding moiety is disclosed. Preferably, the integrin receptor binding moiety is a peptide, and the compositions can be used to deliver DNA to a cell where it will be expressed, for example, to treat a condition by gene therapy. In a preferred embodiment, the integrin receptor binding moiety comprises a peptide, in particular a cyclic peptide, comprising the sequence RGD. In a particularly preferred embodiment, the peptide comprises the sequence GGCRGDMFGC. Cyclic configuration in this sequence is imposed by virtue of the presence of two cysteine residues which can form a disulphide bond.

This is a Continuation of PCT application PCT/GB95/02706, filed Nov. 17,1995.

The present invention relates to compositions comprising DNA associatedwith a polycation entity which is itself linked to an integrin specificbinding moiety. The compositions of the invention can be used to deliverDNA to cells for internalisation and expression therein. In particular,therefore, the invention relates to methods of obtaining gene expressionin cells to overcome genetic deficiencies.

In recent years, with the continuing identification of specific genesresponsible for certain disease conditions, the concept of "genetherapy" has attracted a great deal of attention. The potential todeliver a new gene, or even part of a gene sequence, to a defective cellin order to correct such an inherent deficiency is an attractive one.There are, of course, inherent problems in such an approach. Forinstance, the DNA must be delivered in a form that will be taken up, orinternalised, by the target cell. Furthermore, the DNA itself must beexpressed effectively in the cell in order to overcome the geneticdeficiency. Inherent in these problems is the additional one that theDNA itself, after having entered the cell, must be protected in some wayto prevent its damage, or even destruction, by, for example, cellularenzymes.

One potential approach to this problem of internalisation, andprotection, of DNA is disclosed in Hart et al (J. Biol. Chem., 269, No176: 12468-12474 (1994)). This approach exploits the presence ofintegrin receptors on cell surfaces for achieving internalisation offilamentous phage. Integrins are a super family of heterodimeric celladhesion molecules that consist of several different α an β subunits.Their cellular function is to mediate the movement, shape and polarityof cells through binding with proteins of the extracelluar matrix. Inaddition, integrins are exploited as receptors for cell entry bypathogenic bacteria, such as Yersinia pseudotuberculosis (Isberg, R.,Science, 252: 934-938 (1991)) and Bordatella pertussis (Relamna et al,Cell, 61: 1375-1382 (1990)).

Hart et al (supra) found that displaying an integrin-binding peptidesequence on the surface of bacteriophage fD particles enabled the phageparticles to be internalised by mammalian cells. However, no effectiveexpression of DNA carried by phage particles was shown.

In addition, there are certain problems associated with the use of suchparticles to deliver DNA in this fashion. Firstly, there is a packagingsize limitation governed by the size of the phage particle itself. Onlygenetic material up to a particular particle size could be delivered inthis fashion. Secondly, the phage itself will only package singlestranded genetic material, and this would not be effectively expressedin a mammalian cell system. Finally, the phage itself consists of otherproteins and is a somewhat "messy" system for delivering DNA. It ispossible that these additional components would have a material effecton whether or not genetic material was expressed.

Other approaches to delivering DNA into mammalian cells are disclosed inWO-A-9418834. Here, DNA was conjugated with a polyelectrolyte to form acomplex which was then inserted into an embryonic cell, a germ cell or agerm precursor cell. This method was disclosed primarily for producingtransgenic animals. The methods disclosed in this document rely oneither microinjection of the complex directly into the germ cell, or byhaving the polycation/DNA complex present in the culture medium andrelying on uptake by the cells.

Cotton et al (PNAS USA, 87: 4033-4037 (1990)) used the naturaliron-delivery protein transferrin, coupled to DNA binding polycationssuch as polylysine or protamine, to deliver DNA into human leukaemiccells. However, they also found that they required the use of otheragents to effect the survival of the transfected DNA or to modulatetransferrin receptor levels so as to increase the internalisation oruptake of the DNA itself. These steps included increasing thetransferring receptor density through treatment of the cells with thecell-permeable ion chelator, desferioxamine, interfering with thesynthesis of heme with succinol acetone treatment or stimulating thedegradation of heme with cobalt chloride treatment. In other words,effective uptake and expression of the DNA could only be achievedthrough the use of "co-factors" or co-treatments.

SUMMARY OF THE INVENTION

Thus, there exists a need for further and better methods of deliveringDNA to a cell such that it will be internalised and expressedefficiently therein, preferably without the need for any otherco-factors or co-treatments, and in a form which is not limited togenetic material of a particular size.

BRIEF DESCRIPTION OF THE FIGURES

The examples refer to the figures in which:

FIG. 1: shows a possible structure for the polycation-integrin receptorbinding moiety/DNA complex.

FIG. 2: shows levels of expression of a luciferase reporter protein incells transformed with a composition of the invention, compared tosuitable controls.

FIG. 3: also shows levels of expression of a luciferase reporter proteinin cells with a composition of the present invention, compared tosuitable controls, but here the composition is different from that usedin respect of FIG. 2.

FIG. 4: shows levels of expression of a luciferase reporter protein incells transformed with various compositions of the invention;

FIG. 5: shows the effect of chloroquine on expression of a luciferasereporter protein in COS-7 cells;

FIG. 6: shows the effect of chloroquine on expression of a luciferasereporter protein in endothelial cells;

FIG. 7: shows the results of expression of a luciferase reporter proteinin COS-7 cells using differing ratios of RGD peptide:DNA, in thepresence and absence of chloroquine;

FIG. 8: shows the results of expression of a luciferase reporter proteinin endothelial cells using differing ratios of RGD peptide:DNA, in thepresence and absence of chloroquine; and

FIG. 9: shows the results of comparing transfection of endothelial cellswith varying ratios of K10 peptide:DNA and of K16 peptide:DNA.

DETAILED DESCRIPTION OF THE INVENTION

The approach taken by the present inventors is to use specificcell-surface integrin receptor binding moieties coupled to a polycationmoiety which will bind to DNA.

The "DNA packages" will then bind to cell surface receptors and beinternalised. It is also surprisingly been found that such an approachresults in efficient expression of DNA so internalised, without the needfor any co-factors or co-treatment. Nevertheless, co-factors can be usedwhere desired. A preferred co-factor is chloroquine or any other factorwhich reduces endosomal degradative activity. The observation ofimproved expression in the presence of chloroquine may be because thepeptide-DNA complex is internalised, at least in part, to endosomalcompartments. Chloroquine is a weak buffer which is purported to preventacidification of endosomal vesicles which limits the activity ofendosomal degradative enzymes. Thus the internalised peptide-DNA complexhas more opportunity to escape the endosome and avoid degradation. Otherfactors which might have a similar beneficial effect include ammoniumchloride, another weak buffer which works like chloroquine; fusogenicpeptides related to the N-terminus of the HA protein of influenza viruswhich mediate active membrane disruption and inactivated adenoviruscapsids which also disrupt the membrane of the endosome.

One advantage of integrin receptor mediated internalisation is thatlarge particles can be internalised, e.g. whole cells.

Thus, in a first aspect, the present invention provides a compositioncomprising DNA associated with a polycation wherein the polycation iscoupled to an integrin receptor binding moiety.

In the present invention, "DNA" means single or double stranded DNA,either as complete coding sequences or parts thereof and, in particular,refers to coding sequences for one or more genes.

"Integrin receptor binding moiety" means any moiety or species capableof specifically binding to integrin receptors found on the surface ofcells. In particular, it refers to integrin receptor binding peptidescapable of binding to integrin receptors.

"Association" of the DNA and the polycation occurs, for example, byvirtue of charge-charge interaction, but other forms of association areequally applicable.

In a preferred embodiment, the integrin receptor binding moietycomprises a peptide, in particular a cyclic peptide, comprising thesequence RGD (SEQ ID NO:1). In a particularly preferred embodiment, thepeptide comprises the sequence GGCRGDMFGC (SEQ ID NO:2). Cyclicconfiguration in this sequence is imposed by virtue of the presence oftwo cysteine residues which can form a disulphide bond.

The compositions of the present invention bind effectively to integrinreceptors found on cell surfaces and are internalised. The DNA is theneffectively expressed by the cell without the need for any otherco-factors being present or the need for any co-treatment. Of course,co-factors or co-treatments can be used in conjunction with the presentinvention to boost expression levels even further.

The polycation moiety can be any suitable polycation capable of forminga complex with DNA. In particular polycations such as polylysine can beused. The number of residues in the polycation can vary from arelatively small number, up to quite long chains, or can be a mixturethereof. For example, polycations of from 3-1000, 3-500 or indeed 3-100residues can be used. In particular, 10-16 cation residues are suitable,particularly 16. In one embodiment of the invention, therefore, thepolycation consists of 10-16 polylysine residues, with 16 lysineresidues being particularly preferred.

It is believed that the polycation "tails" of the compositions of theinvention associate with the DNA to be delivered, effectively forming a"package" with the integrin receptor binding moieties on the outside.The DNA composition can then bind to an integrin receptor on the cellsurface and be internalised. The polycation may then act to protect theDNA from the cell's internal enzyme systems, enabling it to beintegrated in the cell's genome and thus expressed.

In a further aspect, the present invention provides a DNA bindingcomposition comprising an integrin receptor binding moiety coupled to apolycation. Preferably the integrin binding moiety is a peptide, asdescribed above. Here coupling may occur to the C-terminus or to theN-terminus of the peptide. In one preferred embodiment, the polycationis polylysine.

This composition can then simply be brought into contact with DNA to"package" the DNA for delivery to a designated cell.

As discussed herein, compositions of the present invention enable theeffective delivery of DNA to cells wherein it is internalised andexpressed efficiently. In this way, genetic deficiencies of particularcell types can be overcome by the delivery and expression of DNAsequences encoding correct or "native" proteins. One example where suchan approach may be effective is in the treatment of cystic fibrosis.Thus, in other aspects, the present invention provides:

(a) the use of a composition of the invention in the manufacture of amedicament for the treatment of prophylaxis of a condition related to agenetic deficiency or modification;

(b) a composition of the invention for use in the treatment orprophylaxis of a condition related to a genetic deficiency ormodification;

(c) a method for the treatment or prophylaxis of a condition related toa genetic deficiency comprising the step of administering to a subject acomposition of the invention;

(d) a method for the transformation of a host cell comprising the stepof bringing together the cell with a composition of the invention; suchmethods find use generally in transfection of cells, particularlymammalian cells;

(e) the use of a composition of the invention in the preparation of amedicament for the treatment or prophylaxis of a condition caused by agenetic deficiency or modification; and

(f) a pharmaceutical formulation comprising a composition of theinvention together with one or more pharmaceutically acceptablecarriers, diluents or excipients.

Preferred features of each aspect of the invention are as for each otheraspect mutatis mutandis.

The invention will now be described by reference to the followingexamples.

EXAMPLE 1 Preparation of a First Polylysine-Integrin Receptor BindingPeptide

The peptide sequence GGCRGDMFGC(K)₁₆ (SEQ ID NO:3) was synthesised asfollows:

(a) the peptide was synthesised on ABI model 431A solid-phase batchpeptide synthesiser using Wang HMP resin and FMOC-cleavage strategy;

(b) the linear peptide was cleaved from the resin using 5 ml of ascavenger mixture (0.75 g phenol, 0.25 ml EDT, 0.5 ml thioanisole, 0.5ml deionised H₂ O, 10 ml TFA), the mixture was stirred for 2 hrs at roomtemperature and was then filtered over sinter into ice-cold MTBE. Thiswas then stored at -18° C. before being spun down, washed with 3×6 mlMTBE before being dried in vacuo and redissolved in H₂ O and freezedried;

(c) cyclisation of the peptide was carried out in 5% AcOH/20% DMSO v/v,buffered to pH 6 by 0.88 NH₃ (aq), with stirring for 24 hrs at roomtemperature. Finally, it was diluted (×3) using deionised water;

(d) purification by ion-exchange chromatography was carried out usingmono-S resin, 50 mM HEPES buffer (pH 7.6) on Pharmacia FPLC system(monitored at 280 nm/UV-Hg lamp);

(e) fractions were assayed for effects on cell cultures;

(f) positive fractions were desalted using P2 Biogel and 0.1% aq TFA;and

(g) further desalting was carried out using reverse-phase chromatographyand 0.1% aq TFA on an FPLC system (monitored at 214 nm/Zn lamp).

EXAMPLE 2 Internalisation and Expression of a Reporter Gene Using thePolylysine-Integrin Receptor Binding Peptide Described in Example 1

5 μg of a luciferase reporter gene plasmid (pGL2 promega) was complexedwith either the RGD-polylysine construct (possible structure of complexis shown in FIG. 1) or an equal concentration of polylysine in 100 μl ofOptimem media (Gibco). The DNA/peptide complexes and also a DNA onlycontrol were then applied to 50% confluent cultured (Caco-2) colonicepithelial cells which were then allowed to express for 48 hours. Thecells were then harvested and the cellular protein analysed forluciferase activity (Relative Light Units). The activity shown in FIG. 2is adjusted to represent activity from 1 mg of cellular protein. In FIG.2 the term "RGD peptide" is used to indicate the polylysine-integrinreceptor binding protein described in Example 1.

EXAMPLE 3 Preparation of a Second Polylysine-Integrin Receptor BindingPeptide

The peptide sequence (K)₁₆ GGCRGDMFGCA (SEQ ID NO:4) was synthesised.This can be done using analogous techniques to those used in respect ofExample 1. This peptide has a similar sequence to that referred to inExample 1, the main difference being that the polylysine region waspresent at the N-terminus rather than the C-terminus.

EXAMPLE 4 Internalisation and Expression of Reporter Gene Using thePolylysine-Integrin Receptor Binding Peptide Described in Example 3

The procedure described in Example 2 was repeated but using thepolylysine-integrin receptor binding peptide described in Example 3instead of the polylysine-integrin receptor binding peptide described inExample 1. Control experiments were also performed. The results areshown in FIG. 3, wherein:

DNA control=5 μg of PGL2 plasmid DNA

PolyK=poly-L-lysine

RGD=newly synthesised RGD-containing peptide linked to polylysine(described in Example 3)

Chlor=100 μM chloroquine

2×RGD+DNA=twice as much as peptide, DNA same (5 μg)

5×RGD+DNA=five times as much peptide.

An indirect comparison of optimal peptide-DNA transfection efficienciesin the absence of chloroquine suggests that the peptide prepared inExample 3 (±6×10⁷ RLU/mg) is more efficient than the peptide prepared inExample 1 (±3.5×10⁵ RLU/mg in FIG. 2) at delivery and expression of theluciferase reporter gene. The addition of chloroquine improvedexpression a further 2 fold approximately, suggesting that endosomaldegradation is limiting the expression levels somewhat. It isinteresting in comparison, however, that, in some circumstances, theefficiency of the transferrin-polylysine receptor-mediated gene deliverysystem was improved by more than 1,000-fold in the presence ofchloroquine. The high level of expression by the RGD-polylysine peptidewithout co-factors and the relatively small improvement in enhancementwith chloroquine is surprising.

EXAMPLE 5 Transfection of Caco-2 Cells with K16 RGD Peptide and Effectof Chloroquine

The polylysine-integrin receptor binding peptide of Example 1 was usedin a repeat of the procedure of Example 2, but using 1 μg of luciferasereporter gene plasmid. The effect of chloroquine was also investigated.The results are shown in FIG. 4. Highest transfection levels wereachieved with 2×RGD peptide and 100 nm chloroquine, these transfectionlevels being approximately 10-fold lower than those achieved withlipofectamine. It can again be seen that chloroquine gave a relativelysmall improvement with 2×RGD peptide+DNA, although with RGD-peptide+DNAthe improvement was greater.

EXAMPLE 6 Effects of Chloroquine on Transfection of COS-7 cells with RGDpeptides

The polylysine-integrin receptor binding peptide of Example 1 was againused. Essentially the methodology of Example 2 was followed with thesubstitution of COS-7 cells, and the use of different concentrations ofchloroquine. The results are shown in FIG. 5. It can be seen that 200 μmchloroquine gave a 4-fold increase of expression, compared with nochloroquine. However, at these levels, cytopathic effects of chloroquinewere apparent.

EXAMPLE 7 Effects of Chloroquine on Transfection of Endothelial Cellswith RGD Peptides

This was a repeat of Example 6 using ECV 304 endothelial cells. Theresults are shown in FIG. 6. For these cells, the highest level ofexpression was obtained in the absence of chloroquine. Althoughincreasing levels of chloroquine restored expression to some extent,complete restoration was not achieved.

EXAMPLE 8 Transfection of COS-7 cells with RGD-Polylysine Peptides

The methodology of Example 2 was repeated using COS-7 cells. In thisexperiment, different ratios of the K16 RGD peptide DNA were used withand without 100 μm chloroquine, with the results shown in FIG. 7. It canbe seen that the optimum ratio was 37.5:1 in both the absence andpresence of chloroquine.

EXAMPLE 9 Transfection of Endothelial Cells with RGD-Polylysine Peptides

This was a repeat of Example 8 using ECV 304 endothelial cells. Theresults are shown in FIG. 8.

EXAMPLE 10 Transfection of Endothelial Cells Comparing K10 and K16 RGDPeptides with Lipofectamine

ECV 304 endothelial cells were transfected with the optimised ratio ofK16 RGD peptide-DNA complex (37.5:1) derived from Example 9. Inaddition, a range of ratios of K10 RGD peptide-DNA complex were alsoinvestigated, as well as transfection using lipofectamine. The resultsare shown in FIG. 9, with the optimised K16 RGD-DNA complex beingz themost efficient.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Arg Gly Asp                                                              1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Gly Gly Cys Arg Gly Asp Met Phe Gly Cys                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Gly Gly Cys Arg Gly Asp Met Phe Gly Cys Ly - #s Lys Lys Lys Lys        Lys                                                                             1               5   - #                10  - #                15              - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys                                              20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys Lys Lys      1               5   - #                10  - #                15               - - Gly Gly Cys Arg Cys Asp Met Phe Gly Cys Al - #a                                      20      - #            25                                        __________________________________________________________________________

We claim:
 1. A composition comprising DNA which encodes a peptide orprotein, said DNA being operatively linked to a transcriptionalregulatory sequence and said DNA being associated with a polycationconsisting of 3-100 lysine residues wherein the polycation is itselfcoupled to an integrin receptor binding protein or peptide.
 2. Thecomposition as claimed in claim 1 wherein the integrin receptor bindingprotein or peptide is a peptide.
 3. The composition of claim 1 whereinsaid integrin receptor binding protein or peptide is a peptide whichcomprises a sequence which comprises the amino acid sequence RGD, SEQ IDNO.
 1. 4. The composition as claimed in claim 1 wherein the integrinreceptor binding peptide or protein comprises the amino acid sequenceRGD, SEQ ID NO.
 1. 5. The composition as claimed in claim 1 wherein theintegrin receptor binding peptide or protein is cyclic.
 6. Thecomposition as claimed in claim 5 wherein the integrin receptor bindingpeptide or protein comprises at least two cysteine residues capable offorming a disulphide bond.
 7. The composition as claimed in claim 6wherein the integrin receptor binding peptide or protein comprises theamino acid sequence GGCRGDMFGC, SEQ ID NO.
 2. 8. The composition asclaimed in claim 1, wherein the polycation consists of 10-16 cationresidues.
 9. The composition as claimed in claim 8 wherein thepolycation consists of 16 cation residues.
 10. The composition asclaimed in claim 9 wherein the ratio of polycation to DNA is 37.5:1. 11.The composition as claimed in claim 1 wherein the DNA is doublestranded.
 12. The composition of claim 1 wherein said DNA encodes agene.
 13. The composition as claimed in claim 11 wherein the DNAcomprises one or more complete gene coding sequences.
 14. Thecomposition as claimed in claim 13 wherein the DNA includes the codingsequence of the cystic fibrosis gene.
 15. The composition according toclaim 1, further comprising a factor which reduces endosomal degradativeactivity.
 16. A DNA binding composition comprising a polycationconsisting of 3-100 lysine residues coupled to an integrin receptorbinding protein or peptide.
 17. The composition as claimed in claim 16wherein the integrin receptor binding protein or peptide is a peptide.18. The composition as claimed in claim 16 wherein the integrin receptorbinding peptide or protein comprises the amino acid sequence RGD, SEQ IDNO.1.
 19. The composition as claimed in claim 16 wherein the integrinreceptor binding peptide or protein is cyclic.
 20. The composition asclaimed in claim 16 wherein the integrin receptor binding peptide orprotein comprises at least two cysteine residues capable of forming adisulphide bond.
 21. The composition as claimed in claim 20 wherein theintegrin receptor binding peptide or protein comprises the amino acidsequence GGCRGDMFGC, SEQ ID NO.
 2. 22. The composition as claimed inclaim 16 wherein the polycation consists of 10-16 cation residues. 23.The composition as claimed in claim 22 wherein the polycation consistsof 16 cation residues.
 24. A method of preparing a composition asdefined in claim 1 which comprises the step of combining said DNA with aDNA binding composition, said DNA binding composition comprising apolycation consisting of 3-100 lysine residues coupled to an integrinreceptor binding protein or peptide.
 25. The method as claimed in claim24 wherein the DNA is double stranded DNA.
 26. The method of claim 24wherein the DNA comprises one or more complete gene coding sequences.27. The method of claim 24 wherein the DNA comprises the coding sequenceof the cystic fibrosis gene.
 28. A method of expressing DNA whichencodes a peptide or protein in a host cell which comprises the step ofcombining in vitro the host cell and a composition as defined inclaim
 1. 29. The method as claimed in claim 28 wherein the host cell isa mammalian cell.
 30. A method for preparing the composition as definedin claim 16 comprising the steps of:(a) synthesising an integrinreceptor binding protein or peptide coupled to a polycation consistingof 3-100 lysine residues; (b) subjecting the integrin receptor bindingprotein or peptide to a cyclisation step; (c) purifying the mixture ofpeptides obtained by ion-exchange chromatography; (d) assaying fractionsfor presence of integrin receptor binding protein or peptide; (e)desalting fractions containing integrin receptor binding protein orpeptide; and (f) further desalting the integrin receptor binding proteinor peptide containing fractions using reverse phase chromatography.